Cestmir Altaner

Adipose Tissue–Derived Human Mesenchymal Stem Cells Mediated Prodrug Cancer Gene Therapy

Human adipose tissue-derived mesenchymal stem cells (AT-MSC) are considered to be a promising source of autologous stem cells in personalized cell-based therapies. Tumor tracking properties of MSC provide an attractive opportunity for targeted transgene delivery into the sites of tumor formation. In the present study, we addressed whether the suicide gene introduction into human AT-MSC could produce a tumor-specific prodrug converting cellular vehicle for targeted chemotherapy. We prepared yeast fusion cytosine deaminase::uracil phosphoribosyltransferase gene-expressing cells [cytosine deaminase (CD)-expressing AT-MSC (CD-AT-MSC)] by retrovirus transduction. We explored their therapeutic potential on a model of human colon cancer in the presence of prodrug 5-fluorocytosine (5-FC). Gene manipulation of human AT-MSC did not sensitize CD-AT-MSC to 5-FC, thus overcoming the inherent disadvantage of suicide effect on cellular vehicle. CD-AT-MSC in combination with 5-FC augmented the bystander effect and selective cytotoxicity on target tumor cells HT-29 in direct coculture in vitro. We confirmed directed migration ability of AT-MSC and CD-AT-MSC toward tumor cells HT-29 in vitro. Moreover, we achieved significant inhibition of s.c. tumor xenograft growth by s.c. or i.v. administered CD-AT-MSC in immunocompromised mice treated with 5-FC. We confirmed the ability of CD-AT-MSC to deliver the CD transgene to the site of tumor formation and mediate strong antitumor effect in vivo. Taken together, these data characterize MSC derived from adipose tissue as suitable delivery vehicles for prodrug converting gene and show their utility for a personalized cell-based targeted cancer gene therapy.

AC-magnetic field controlled drug release from magnetoliposomes: design of a method for site-specific chemotherapy

Large unilamellar magnetoliposomes (MLs) with encapsulated doxorubicin (DOX) (anticancer drug) were prepared by reverse-phase evaporation. They were exposed to an alternating magnetic field with a frequency of 3.5 MHz and an induction of 1.5 mT produced in three-turn pancake coil. The results showed that magnetoliposomes could be specifically heated to 42 degrees C (phase transition temperature of a used lipid) in a few minutes and during this, the encapsulated doxorubicin is massively released.

Cytosine deaminase expressing human mesenchymal stem cells mediated tumour regression in melanoma bearing mice.

Previously, we validated capability of human adipose tissue‐derived mesenchymal stem cells (AT‐MSC) to serve as cellular vehicles for gene‐directed enzyme prodrug molecular chemotherapy. Yeast fusion cytosine deaminase : uracil phosphoribosyltransferase expressing AT‐MSC (CDy‐AT‐MSC) combined with systemic 5‐fluorocytosine (5FC) significantly inhibited growth of human colon cancer xenografts. We aimed to determine the cytotoxic efficiency to other tumour cells both in vitro and in vivo.

Tumor cell behaviour modulation by mesenchymal stromal cells

BackgroundHuman mesenchymal stromal cells (MSC) hold a promise for future cell-based therapies due to their immunomodulatory properties and/or secretory activity. Nevertheless non-neoplastic tumor compartment could also originate from MSC. We aimed to show whether multipotent MSC derived from human adipose tissue (AT-MSC) could create tumor cell-protective milieu and affect tumor cell behaviour in vitro and in vivo.ResultsHere we have demonstrated tumor-promoting effect of AT-MSC on human melanoma A375 cells. AT-MSC coinjection mediated abrogation of tumor latency and supported subcutaneous xenotransplant growth from very low melanoma cell doses. Tumor incidence was also significantly increased by AT-MSC-derived soluble factors. AT-MSC supported proliferation, suppressed apoptosis and modulated melanoma cell responses to cytotoxic drugs in vitro. Expression and multiplex cytokine assays confirmed synergistic increase in VEGF that contributed to the AT-MSC-mediated support of A375 xenotransplant growth. Production of G-CSF and other factors implicated in formation of supportive proinflammatory tumor cell microenvironment was also confirmed. SDF-1α/CXCR4 signalling contributed to tumor-promoting effect of systemic AT-MSC administration on A375 xenotransplants. However, no support was observed for human glioblastoma cells 8MGBA co-injected along with AT-MSC that did not sustain tumor xenotransplant growth in vivo. Tumor-inhibiting response could be attributed to the synergistic action of multiple cytokines produced by AT-MSC on glioblastoma cells.ConclusionsHerein we provide experimental evidence for MSC-mediated protective effect on melanoma A375 cells under nutrient-limiting and hostile environmental conditions resulting from mutual crosstalk between neoplastic and non-malignant cells. This tumor-favouring effect was not observed for the glioblastoma cells 8MGBA. Collectively, our data further strengthen the need for unravelling mechanisms underlying MSC-mediated modulation of tumor behaviour for possible future MSC clinical use in the context of malignant disease.

HSV-tk expressing mesenchymal stem cells exert bystander effect on human glioblastoma cells

Previously we have reported adipose-tissue derived human mesenchymal stem cells (AT-MSC) as cellular delivery vehicles for tumor-targeted cancer gene therapy. In this report we aimed to determine whether Herpes simplex virus - thymidine kinase (HSV-tk) expressing AT-MSC (TK-MSC) could exert cytotoxic effect on tumor cells upon treatment with prodrug ganciclovir (GCV). Direct co-cultures of human glioblastoma cells 8-MG-BA, 42-MG-BA and U-118 MG with TK-MSC/GCV resulted in substantial viability decrease in vitro. This therapeutic paradigm was most efficient against 8-MG-BA glioblastoma cells exhibiting cytotoxicity (>50%) in the presence of TK-MSC and 0.1microM GCV. Rapid apoptosis induction in three glioblastoma cell lines and TK-MSC demonstrated both bystander cytotoxic effect on tumor cells and GCV conversion-mediated suicide effect on TK-MSC. Furthermore, we were able to demonstrate formation of gap junctions between AT-MSC and human glioblastoma cells as a mechanism contributing to bystander cytotoxicity. Inability of human HeLa and MCF7 to form gap junctions with AT-MSC rendered these cell refractory to the TK-MSC/GCV mediated cytotoxicity. Gap junction intercellular communication (GJIC) capability of AT-MSC with tumor cells further supports the exploitation of mesenchymal stem cells for approaches relying on the bystander effect. Biological consequences of these capabilities remain to be further explored.

Adipose Tissue–derived Mesenchymal Stem Cells Expressing Prodrug-converting Enzyme Inhibit Human Prostate Tumor Growth

The ability of human adipose tissue-derived mesenchymal stem cells (AT-MSCs), engineered to express the suicide gene cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT), to convert the relatively nontoxic 5-fluorocytosine (5-FC) into the highly toxic antitumor 5-fluorouracil (5-FU) together with their ability to track and engraft into tumors and micrometastases makes these cells an attractive tool to activate prodrugs directly within the tumor mass. In this study, we tested the feasibility and efficacy of these therapeutic cells to function as cellular vehicles of prodrug-activating enzymes in prostate cancer (PC) therapy. In in vitro migration experiments we have shown that therapeutic AT-MSCs migrated to all the prostate cell lines tested. In a pilot preclinical study, we observed that coinjections of human bone metastatic PC cells along with the transduced AT-MSCs into nude mice treated with 5-FC induced a complete tumor regression in a dose dependent manner or did not even allow the establishment of the tumor. More importantly, we also demonstrated that the therapeutic cells were effective in significantly inhibiting PC tumor growth after intravenous administration that is a key requisite for any clinical application of gene-directed enzyme prodrug therapies.

Prodrug cancer gene therapy

There is no effective treatment for late stage and metastatic cancers of colorectal, prostate, pancreatic, breast, glioblastoma and melanoma cancers. Novel treatment modalities are needed for these late stage patients because cytotoxic chemotherapy offers only palliation, usually accompanied with systemic toxicities and poor quality of life. Gene directed enzyme prodrug therapy (GDEPT), which concentrates the cytotoxic effect in the tumor site may be one alternative. This review provides an explanation of the GDEPT principle, focusing on the development, application and potential of various GDEPTs. Current gene therapy limitations are in efficient expression of the therapeutic gene and in tumor-specific targeting. Therefore, the current status of research related to the enhancement of in situ GDEPT delivery and tumor-specific targeting of vectors is assessed. Finally, GDEPT versions of stem cell based gene therapy as another potential treatment modality for progressed tumors and metastases are discussed. Combinations of traditional, targeted, and stem cell directed gene therapy could significantly advance the treatment of cancer.

In Vitro Analysis of Cisplatin Functionalized Magnetic Nanoparticles in Combined Cancer Chemotherapy and Electromagnetic Hyperthermia

A novel platform has been developed for combined cancer chemotherapy and hyperthermia based on iron oxide magnetic nanoparticles functionalized with cis-diamminedichloroplatinum(II) (cisplatin). The capabilities of this system for heating and controlled drug release were investigated, and the system was tested in vitro by the treatment of BP6 rat sarcoma cells, where we demonstrated a synergism between the effects of cisplatin-targetMAG nanoparticles and the application of electromagnetic field.

Stem Cell Based Cancer Gene Therapy

The attractiveness of prodrug cancer gene therapy by stem cells targeted to tumors lies in activating the prodrug directly within the tumor mass, thus avoiding systemic toxicity. Suicide gene therapy using genetically engineered mesenchymal stem cells has the advantage of being safe, because prodrug administration not only eliminates tumor cells but consequently kills the more resistant therapeutic stem cells as well. This review provides an explanation of the stem cell-targeted prodrug cancer gene therapy principle, with focus on the choice of prodrug, properties of bone marrow and adipose tissue-derived mesenchymal stem and neural stem cells as well as the mechanisms of their tumor homing ability. Therapeutic achievements of the cytosine deaminase/5-fluorocytosine prodrug system and Herpes simplex virus thymidine kinase/ganciclovir are discussed. In addition, delivery of immunostimulatory cytokines, apoptosis inducing genes, nanoparticles and antiangiogenic proteins by stem cells to tumors and metastases is discussed as a promising approach for antitumor therapy. Combinations of traditional, targeted and stem cell-directed gene therapy could significantly advance the treatment of cancer.

Human adipose tissue‐derived mesenchymal stem cells expressing yeast cytosinedeaminase::uracil phosphoribosyltransferase inhibit intracerebral rat glioblastoma

Prodrug cancer gene therapy by mesenchymal stem cells (MSCs) targeted to tumors represents an attractive tool to activate prodrugs directly within the tumor mass, thus avoiding systemic toxicity. In this study, we tested the feasibility and efficacy of human adipose tissue‐derived MSCs, engineered to express the suicide gene cytosine deaminase::uracil phosphoribosyltransferase to treat intracranial rat C6 glioblastoma. Experiments were designed to simulate conditions of future clinical application for high‐grade glioblastoma therapy by direct injections of therapeutic stem cells into tumor. We demonstrated that genetically modified therapeutic stem cells still have the tumor tropism when injected to a distant intracranial site and effectively inhibited glioblastoma growth after 5‐fluorocytosine (5‐FC) therapy. Coadministration of C6 cells and therapeutic stem cells with delayed 5‐FC therapy improved the survival in a therapeutic stem cell dose‐dependent manner and induced complete tumor regression in a significant number of animals. Continuous intracerebroventricular delivery of 5‐FC using osmotic pump reduced the dose of prodrug required for the same therapeutic effect, and along with repeated administration of therapeutic stem cells increased the survival time. Intracerebral injection of therapeutic stem cells and treatment with 5‐FC did not show any detectable adverse effects. Results support the arguments to begin clinical studies for treatment of high‐grade brain tumors.